Analysis of the biosynthesis of HLA-DR glycoproteins in human malignant melanoma cell lines.
Human malignant melanoma cell line SK-MEL-37 expresses HLA-DR antigens having a characteristic 2-chain structure, with heavy chains (alpha) of approximately 32,000 daltons and light chains (beta) of approximately 28,000 daltons. Nonequilibrium pH-gradient electrophoresis (NEPHGE) on HLA-DR immunoprecipitated by rabbit anti-HLA-DR sera from [35S]-methionine-labeled, pulse-chased cells showed 2 closely spaced heavy-chain components (1, 2) and 4 light-chain spots (3-6). From nonchased samples, numerous more-basic components running in the heavy chain region were also precipitated. In particular, a very basic, 30,000 dalton component (pl approximately 8.5) was prominent (component 7); this spot probably corresponds to the invariant (Ii) peptide previously demonstrated in lymphoid cell HLA-DR precipitates (9) and the M-1 peptide of Shackelford and Strominger (10). None of these components (alpha, beta, or component 7) was precipitated from extracts of HLA-DR-negative melanoma cells. Pulse-chase experiments and the use of different labeled sugar precursors showed that component 7 is a partially glycosylated intracellular precursor, possibly of the HLA-DR alpha-chain. None of the immunoprecipitates, even from cells pulse-labeled for only 15 min, contained a peptide migrating in the 55,000 to 60,000 m.w. region. It was concluded that melanoma HLA-DR is not synthesized via a polyprotein precursor. In contrast to these results, obtained with rabbit anti-HLA-DR sera, a mouse monoclonal anti-HLA-DR was found to precipitate only biosynthetically completed alpha (1, 2) and beta (3-6) chains.
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D008545:Melanoma / Q000032:analysis*; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D011817:Rabbits
D000906:Antibodies; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D000949:Histocompatibility Antigens Class II; D007106:Immune Sera; D008715:Methionine; D000469:Alkaline Phosphatase; D009439:Neuraminidase